Plants synthesize an overwhelming variety of triterpene saponins with an enormous range of biological activities relevant for the pharmaceutical and chemical industry (e.g., additives to foods and cosmetics). Interest in triterpenoid saponins and its precursors has increased recently because of data showing their diverse biological activities and beneficial properties, which include antifungal, antibacterial, antiviral, antitumor, molluscicidal, insecticidal, and antifeedant activities (Suzuki et al. 2002; Sparg et al. 2004; Huhman et al. 2005). Saponins are synthesized by multiple glycosylations of sapogenin building blocks, which, in turn, are produced by multiple modifications (e.g., hydroxylations) of basic sapogenin backbones such as β-amyrin, lupeol, and dammarenediol. These diverse backbones are generated by specific cyclizations of 2,3-oxidosqualene, which is also an intermediate in the synthesis of membrane sterols. As an illustration, more than seventy saponins have been identified in the model legume, M. truncatula (Huhman and Sumner 2002; Pollier et al. 2011), the core of this diversity being centralized in a few aglycones (sapogenins). Also, these precursor sapogenins are very valuable compounds and are important starter molecules for further synthetic modifications. For example, the naturally occurring triterpenoid sapogenin oleanolic acid and its derivatives possess several promising pharmacological activities, such as hepato-protective effects, and anti-inflammatory, antioxidant, or anticancer activities (Pollier and Goossens 2012).
The first committed step in triterpenoid saponin biosynthesis is the cyclization of 2,3-oxidosqualene (FIG. 1). This reaction is catalyzed by specific oxidosqualene cyclases (OSCs), including β-amyrin synthase (bAS; EC 5.4.99.-), and has been functionally characterized in several plants (Kushiro et al. 1998, Herrera et al. 1998, Iturbe-Ormaetxe et al. 2003, Morita et al. 2000, Suzuki et al. 2002). Then, the action of oxidative enzymes (typically cytochrome P450 monooxygenases or CYPs) and glycosyltransferases convert β-amyrin to various triterpene saponins in different plant species. For example, subsequent modifications that impart functional properties and diversify the basic triterpenoid backbone include the addition of small functional groups, including hydroxyl, keto, aldehyde, and carboxyl moieties, generally followed by glycosylation reactions (Augustin et al. 2011). To date, a number of CYPs that use β-amyrin as a substrate have been identified in dicotyledonous plants, whereas just one (CYP51H10) has been identified in monocots.
Present availability of saponins and sapogenins depends on their extractability from plants and is often uneconomical and inefficient. Often, laborious extraction schemes have to be developed for each specific metabolite of interest and a steady supply of sufficient amounts of specific sapo(ge)nins from plants that accumulate mixtures of structurally related compounds is not feasible. Synthetic chemistry mainly attempts to address these issues by chemically linking desired side chains to extracted sapogenins, as was done for oleanolic acid. However, the structural complexity of the sapogenins hampers chemical synthesis and the availability of corresponding sapogenins forms a major bottleneck.
The culture of plant cells has been explored since the 1960s as a viable alternative for the production of complex phytochemicals of industrial interest. For example, the use of large-scale plant cell cultures in bioreactors for the production of alkaloids has been extensively studied (Verpoorte et al. 1999). Despite the promising features and developments, the production of plant-derived pharmaceuticals by plant cell cultures has not been fully commercially exploited. The main reasons for this reluctance shown by industry to produce phytochemicals by means of cell cultures, compared to the conventional extraction of whole plant material, are economical ones based on the slow growth and the low production levels of phytochemicals by such plant cell cultures. Important causes are the toxicity of such compounds to the plant cell, and the role of catabolism of these compounds. Another important problem is that many phytochemicals, such as the triterpene saponins and its precursors, are mostly retained intracellularly, complicating the downstream processing and purification. Another important problem is that for many phytochemicals, the precursors or intermediates in the pathway do not accumulate or only in trace amounts, because they are readily converted by the downstream enzymes.
Biotechnological production of either complete saponins, or of sapogenin pathway intermediates that are not readily accessible, may circumvent the limitation of natural sapo(ge)nin availability. However, circumvention of laborious and uneconomical extraction procedures for industrial production from plants is also hampered by lack of knowledge and availability of genes in saponin biosynthesis. As a consequence, although triterpene synthases have been expressed in microbial hosts such as Saccharomyces cerevisiae, there has been little effort made so far to engineer the metabolism of a microbial host for enhanced production of triterpenes. By contrast, there have been many considerable efforts to engineer microbes for higher production of mono-, sesqui- and diterpenes. Notably, triterpene production may not be as amenable to engineering efforts as the volatile sesquiterpenes and monoterpenes that readily diffuse out of the cell.
Therefore, a need exists for the cost-effective biotechnological production of high value sapo(ge)nins or other triterpene building blocks in a convenient host cell.